This application is the 35 USC 371 national stage of International Application PCT/IT99/00289 filed on Sep. 15 1999, which designated the United States of America.
The present invention relates to the chemical analysis and more particularly a method of determining the antioxidant power of an organic or inorganic liquid and/or solution by using bathocuproine. According to the invention, the antioxidant power of an organic or inorganic liquid and/or solution is determined by causing it to enter into competition with bathocuproine in a copper sulphate solution. The results compared with the reducing action of uric acid in water solution or xcex1-tocopherol in oil solution with known concentration give values expressed in xcexcmol/litre.
The method used at present for determining the antioxidant power of a sample valid only in water solutions provides the steps of:
providing a solution of ABTS (2,2xe2x80x2-azino-di-3-ethylbenztiazoline sulphonate) with matamioglobine which is a composition stable only 8 hours at room temperature or 48 hours at +2/+8xc2x0 C.; and
causing such solution to enter into competition with the sample to be analyzed in a hydrogen peroxide solution.
The invention seeks to provide a method of determining the antioxidant power of a liquid and/or solution by using a marker consisting of a stable substance and by providing values expressed in xcexcmol/litre. It is known that bathocuproine (BC) forms stable complexes with monovalent Cu. Such a reaction is specific for monovalent Cu(I) and not for divalent CU(II). CU(II) in solution may be reduced to Cu(I) by a number of reducing compositions belonging to a class of compositions consisting prevailingly of both liposoluble (tocopherols, carotenoids, etc.) and water soluble (ascorbic acid, uric acid, bilirubin, etc.) non-enzymatic antioxidants.
For instance, Cu (I) formation was monitored using the bathocuproine as specific CU(I) chelator in BIOCHEMICAL JOURNAL , Vol. 322, n.2 pages 425-433, 1997 R. P. Patel :reduction of Cu(II) by lipid hydroperoxide: implications for the copper-dependent oxidation of low-density lipoproteinxe2x80x9d.
The experiments reported in the overmentioned document exclusively demonstrate that PC liposomes enriched in lipid hydroperoxides (they are not antioxidants) are able to reduce Cu++ to Cu+. This is due to reducing activity of lipid hydroperoxides, but it has nothing to do with any antioxidant activity.
According to the present invention, it is shown that, when the reaction occurs in a bathocuproine (BC) buffer, the complex being formed is characterized by the concentration of the reducing agents and then, by good approximation, of the antioxidants present in the system. The quantitative analysis of such a reaction can be easily made by spectrophotometry at 480 nm both by macro- and micromethods with the use of a number of reducing standard compositions with known concentrations.
One feature of the present invention is the determination of the antioxidant power of the sample (plasma, serum, other biological, non-biological liquids) by adding copper sulphate and measuring Cu(I) ion formed from the reduction of CU (II) ion by all of the present antioxidant substances.
A second peculiar feature of the invention is the detection of Cu(I) ion made by measuring the quantity of Cu(I)-bathocuproine (BC) complex, the latter chelating agent being pure in oil solutions and showing the disulphonate form in water solutions. Such (stable) complex has a typical spectrum of absorption with a maximum at 480-490 nm. The values obtained are quantified by comparison with a standard curve provided by samples having a known concentration of uric acid used as typical reducing agent in water solutions, and xcex1-tocopherol in oil solutions.